Abscission is the process that causes the shedding of a range of plant parts, including leaves, flowers and fruit. The process occurs at precise sites and involves coordinated cell wall breakdown. Associated with cell separation is an increase in the activity of several hydrolytic enzymes including .beta.-1,4-glucanase (cellulase, EC 3.1.2.4) and polygalacturonase (PG EC 3.2.1.15).
The process of pod dehiscence, or shatter as it is commonly termed, in oilseed rape (Brassica napus) and other crops shares a number of features with abscission. Degradation and separation of cell walls occurs along a discrete layer of cells, termed the dehiscence zone, and a localised increase in the activity of cellulase has been reported prior to the onset of dehiscence (Meakin and Roberts J. Exp. Bot. 41(229) 995-1002 (1990) and J. Exp. Bot. 41(229) 1003-1011 (1990)). This process is agronomically important because it may result in the premature shedding of seed before the crop can be harvested. Adverse weather conditions can exacerbate the process resulting in a greater than 50% loss of seed. This loss of seed not only has a dramatic effect on yield but also results in the emergence of the crop as a weed in the subsequent growing season.
Attempts to solve this problem over the last 20 years have focused on the breeding of shatter-resistant varieties. The most commonly used method is by trying to introduce germplasm from related species by interspecific hybridisation. Related species such as B. nigra, B. juncea and B. campestris have been used for this purpose but resulting plants from these crosses are frequently sterile and lose favourable characteristics which have to be regained by back crossing. This is both time consuming and laborious. The interspecific hybridisation strategy also has to cope with transferring two or more genes which are recessive in action into each of the breeding lines. Indeed, even within B. campestris, different genetic backgrounds have revealed different numbers of genes to be important in shatter resistance. This has necessitated breeders performing test crosses at each generation during the attempt to produce elite material. These difficulties have been compounded by the fact that shattering is a difficult and time-consuming trait to assess in the field. All these factors may account for the fact that the conventional breeding approach has made no progress over the last twenty years.
Other methods employed to try and alleviate the problem include chemicals, in the form of desiccants and pod sealants. The most widely used method to try and prevent seed loss is the mechanical technique of swathing in order to get uniform desiccation of the crop and reduce shattering by wind which occurs in the upright crop.
This invention takes a completely different approach to solve the problem of dehiscence: it involves the use of recombinant DNA technology. In 1988, when plant biotechnology had reached an age of some considerable sophistication, Roberts and Taylor speculated:
By regulating cell separation at abscission sites, it may be possible . . . to also influence related processes such as pod dehiscence. (Proceedings of the Symposium on the Physiology of Fruit Drop, Ripening, Storage and Post-Harvest Processing of Fruits, Turin, 3-4 October 1988, pp 24-33). PA1 (i) a transgenic plant which includes a nucleic acid sequence as defined herein; PA1 (ii) an oilseed rape plant which is shatter resistant; PA1 (iii) a plant cell which includes a nucleic acid sequence as defined herein; PA1 (iv) propagating material derived from transgenic plants of the invention; PA1 (v) seeds derived from transgenic plants of the invention; and PA1 (vi) a method of regulating dehiscence, which comprises the step of transforming or transfecting propagating material from a plant with a nucleic acid sequence as defined herein.
However, without any indication of which genes may be involved in such processes, this exhortation did little to enable the art to address the problem at the cell or genetic level.
WO94/23043 discloses nucleic acid sequences encoding proteins involved in plant abscission or dehiscence. However this disclosure does not include any discussion of polygalacturonase's role in abscission or dehiscence.
Meakin and Roberts (supra) reported that there was no correlation between the timing of dehiscence and the activity of the pectin degrading enzyme, polygalacturonase (PG). Although they stated that it was not possible to discount a role for PG in pod shatter, their work did not provide any evidence that activity of the enzyme was related to timing of dehiscence.